Differentiating Oligodendrocytes NSC
| Title | Differentiating Neural Stem Cells into Oligodendrocytes |
| Date Submitted | May 5, 2012 |
| Submitted by - | Efthymiou, Anastasia - anastasia.efthymiou@nih.gov |
| Adapted from - | Gibco Protocol |
| Contributors - | Efthymiou, Anastasia |
| Affiliation(s) - | NIH CRM - NIAMS – Laboratory of Stem Cell Biology |
Introduction:
Oligodendrocyte stained for PLP (green) and an intracellular marker (red) - Prof. Klaus-Armin Nave, Max-Planck-Institute for Experimental Medicine.
Protocol:
Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. If the cells reach 90% confluency, it might be necessary to split the cells at a 1:2 ratio. However, do not split the cells once they reach day 9-10 of differentiation when they can get damaged during the passaging process.
- Plate the NSCs on a polyornithine and laminin-coated culture dish in complete StemPro NSC SFM at 2.5 × 10^4 or 5 × 10^4 cells/cm2.
- After 2 days, change the medium to oligodendrocyte differentiation medium. Change the spent medium every 3 to 4 days.
Materials:
Polyornithine and laminin-coated culture dish
StemPro NSC SFM
Oligodendrocyte differentiation medium
StemPro NSC SFM Complete Media
| Component | Final concentration | Amount |
| KnockOutTM D-MEM/F-12 | 1X | 97 mL |
| GlutaMAXTM-I Supplement | 2 mM | 1 mL |
| bFGF (prep as 100 µg/mL stock) | 20 ng/mL | 20 µL |
| EGF (prep as 100 µg/mL stock) | 20 ng/mL | 20 µL |
| StemPro® Neural Supplement | 2% | 2 mL |
| Component | Final concentration | Amount |
| Neurobasal® Medium | 1X | 97 mL |
| B-27® Serum-Free Supplement | 2% | 2 mL |
| GlutaMAXTM-I Supplement | 2 mM | 1 mL |
| T3 | 30 ng/mL | 0.1 mL |
